Global Change of Gene Expression and Cell Physiology in YidC-Depleted Escherichia coli

JOURNAL OF BACTERIOLOGY, Apr. 2010, p. 2193–2209 Vol. 192, No. 80021-9193/10/$12.00 doi:10.1128/JB.00484-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Global Change of Gene Expression and Cell Physiology inYidC-Depleted Escherichia coli�†

Peng Wang,1 Andreas Kuhn,2 and Ross E. Dalbey1*The Ohio State University, Department of Chemistry, 100 West 18th Avenue, Columbus, Ohio 43210,1 and

Institute of Microbiology and Molecular Biology, University of Hohenheim, 70599 Stuttgart, Germany2

Received 8 April 2009/Accepted 23 December 2009

YidC depletion affects membrane protein insertion and leads to a defect in the growth of the Escherichia colicell. We analyzed global changes in gene expression upon YidC depletion to determine the importance of YidCfor cellular functions using a gene chip method to compare the transcriptomes of JS71 (control) and JS7131(yidC depletion strain). Of the more than 4,300 genes identified, 163 were upregulated and 99 were downregu-lated upon YidC depletion, including genes which are responsible for DNA/RNA repair; energy metabolism;various transporters, proteases and chaperones; stress response; and translation and transcription functions.Real-time PCR was performed on selected genes to confirm the results. Specifically, we found upregulation ofthe genes encoding the energy transduction proteins F1Fo ATP synthase and cytochrome bo3 oxidase due toperturbation in assembly when YidC was depleted. We also determined that the high-level induction of thePspA stress protein under YidC depletion conditions is roughly 10-fold higher than the activation due to theaddition of protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), which dissipates the protonmotive force. In addition, the gene chip data reveal the Cpx stress pathway is activated upon YidC depletion.The data show the broad physiological contribution of YidC to the bacterial cell and the considerableramification to the cell when it is depleted.

According to genomic and proteomic analyses, approxi-mately 20% of proteins encoded by the Escherichia coli ge-nome are membrane proteins (4, 14), and these membraneproteins play important roles in energy metabolism, membranetransport of molecules, stress response, and other biologicalprocesses. All membrane proteins must be inserted and cor-rectly folded in the membrane in order to function properly.Bacterial cells utilize specialized insertion machineries to fa-cilitate these processes. In E. coli, membrane proteins areprimarily inserted by the SecYEG translocase and YidC inser-tase (43).

The YidC insertase has been shown to be critical for theinsertion of several Sec-independent proteins, such as the M13procoat protein and F1Fo ATP synthase subunit c (2, 28, 29, 37,38, 45). In addition to working independently, YidC was foundto cooperate with the SecYEG translocase in catalyzing themembrane integration of F1Fo ATP synthase subunit a andcytochrome bo oxidase subunit II (CyoA) (1, 8, 36, 44). Theseresults explain why depletion of YidC has a marked effect onthe assembly of the cytochrome bo oxidase and F1Fo ATPsynthase (39). YidC may also function as a chaperone for thefolding of polytopic membrane proteins such as LacY (17) andMalF (41) and is required for the assembly of the maltosetransport complex (41).

Recently, it was reported that YidC is present within a large-molecular-weight complex that includes the membrane pro-

tease, FtsH, a protease capable of degrading misassembledmembrane proteins (35). The hypothesis is that this complexmay function in the quality control of membrane proteins, withYidC functioning as a chaperone to fold membrane proteinsand FtsH-degrading proteins that have not obtained theirproper membrane conformation.

To better understand the importance of YidC to cell phys-iology, high-throughput methods such as gene chip and pro-teomic experiments can be performed. Such tools can helpscientists understand the protein function and the cellular roleof the protein by examining the global changes that occur inthe transcriptome and the protein level in a cell when theprotein under study is eliminated or reduced.

In this paper, we present a comprehensive analysis of theeffects of YidC depletion in E. coli using the well-establishedJS71 and JS7131 strains. JS71 was derived from MC1060 andhas 2 copies of yidC: the endogenous gene and one copy underthe control of the arabinose-inducible pBAD promoter (28).JS7131 was constructed from JS71 where the original yidCgene on the chromosome was disrupted by a deletion and acomplementary copy of yidC under the control of an araBADpromoter was incorporated into the chromosome. Thereby, theexpression of YidC in JS7131 can be induced with arabinoseand repressed with glucose (28). Samples were collected 5 hafter the addition of glucose, when the growth defect wasevident, to examine primary effects from the absence of YidCand secondary effects from the absence of YidC’s substrates.

A gene chip and two-dimensional (2D) gel electrophoresismethod was utilized to analyze the change of the gene expres-sion pattern and protein pattern, respectively, when YidC wasdepleted from the cell. Real-time PCR and Western blot anal-ysis were used to confirm the microarray and proteomic anal-ysis studies, respectively. The growth, phenotype, and motility

* Corresponding author. Mailing address: The Ohio State Univer-sity, Department of Chemistry, 100 West 18th Avenue, Columbus, OH43210. Phone: (614) 292-2384. Fax: (614) 292-1532. E-mail: [email protected].

† Supplemental material for this article may be found at http://jb.asm.org/.

� Published ahead of print on 8 January 2010.

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of the cells were also analyzed. This study provides importantinformation on how the bacterial cell responds to a defect inmembrane protein biogenesis. It is also the first study thatdescribes the effect of eliminating YidC function on the level ofthe transcriptome and proteome and on cell physiology.

MATERIALS AND METHODS

Bacterial strains and growth conditions. The E. coli JS71 and JS7131 strains(28) are from our laboratory collection. JS71 was used as the control strain andJS7131 was used as the experimental strain in this study. Starter cultures for bothstrains were grown overnight in LB medium from single colonies at 37°C, and0.2% arabinose was added to JS7131. Before back dilution, the JS7131 culturewas washed with LB medium three times to remove all traces of arabinose.Overnight cultures were then diluted 1 to 50 into LB media plus 0.2% glucose.After dilution, both JS71 and JS7131 were vigorously shaken and grown for 5 hbefore cells were harvested.

Growth curve and microscopy. Cells were grown as described above with 3 mlof cultures of each strain to obtain the growth curve. The growth of the cells wasmonitored by measuring the optical density at 600 nm (OD600) at 1 h after backdilution and then each hour thereafter for 12 h. Cell cultures were grown inLB-glucose medium for 5 h for both JS71 (YidC expression condition) andJS7131 (YidC depletion condition) strains and stained using 0.3% methyleneblue for better visualization of the cells under the microscope. Pictures weretaken using a Nikon FDX-35 digital camera attached to the Nikon Eclipse E800microscope at a �100 magnification.

Cell motility assay. LB motility agar plates were used to compare the motilityof JS71 expressing normal YidC levels and JS7131 after YidC depletion. The LBmotility agar was made with 2.5% LB broth Miller (Novagen) and 0.3% agar.Glucose (0.2%) and arabinose (0.002%) were added to the plates to ensure thatJS7131 survived the subsequent long-time incubation, while the expression ofYidC protein was kept at a low level. After 5 h of YidC depletion in liquidculture, equal amounts of JS71 and JS7131 cells (normalized using a microscopeand counting chamber) were added to the LB motility agar plates and the plateswere incubated at 37°C for 24 h. The diameters of the swimming zones formedby the motile bacteria were measured to determine their motility.

RNA and cDNA preparation for gene chip experiments. Cells were grown in10-ml cultures of each strain for RNA preparation. Cells were mixed with 2volumes of RNAprotect bacterial reagent (Qiagen) to stabilize mRNA. AnRNeasy minikit was used to purify mRNA followed by on column DNase diges-tion. The quality of the purified total RNA was checked by measuring OD260 andOD280. Samples were either stored at �20°C or used immediately for cDNApreparation.

cDNA was synthesized using high-capacity cDNA reverse transcription kits(Applied Biosystems). RNase inhibitor was added to prevent the degradation ofRNA during PCR cycles. The reaction was carried out in an MJ ResearchMinicycler. The conditions were 25°C for 10 min, 37°C for 120 min, and 85°C for5 s for 30 cycles. cDNA was then purified from the reaction mix by using theQIAquick PCR purification kit. The concentration of cDNA was measured atOD260 using an extinction coefficient of 35 ng/�l per OD unit.

Gene chip experiments and data analysis. The gene chip experiments and rawdata analysis were performed in the Biomedical Genomics core at the ColumbusChildren’s Hospital. Total RNA was checked with a capillary electrophoresisBioanalyzer 2100 (Agilent) for integrity and measured with a Nanodrop 1000spectrophotometer (Nanodrop, Wilmington, DE) for quantification. Biotin-la-beled cDNA was then synthesized from total RNA using the Ovation biotinRNA amplification and labeling system (NuGen Technologies, Inc., San Carlos,CA) following the manufacturer’s protocol. Twenty-five nanograms of total RNAwas used for synthesizing the first-strand cDNA, followed by double-strandedcDNA amplification. The amplified cDNA was purified using a NucleoSpinextract kit (BD Clontech). Fragmentation and biotin labeling were achievedusing NuGEN FL-Ovation cDNA biotin module V2 (NuGen Technologies, Inc.,San Carlos, CA). The quality and quantity of the fragmented/biotinylated cDNAwere analyzed using the Nanodrop spectrophotometer and Bioanalyzer 2100,respectively.

E. coli Genome 2.0 GeneChips (Affymetrix, Inc.) were used for this experi-ment, with approximately 10,000 probe sets for all 20,366 genes that are presentin four strains of E. coli. For each gene chip, 2 �g of cDNA was applied. Thehybridization process was carried out at 45°C for 16 h. The gene chip was thenwashed and stained in a Fluidics Station 450 machine (Affymetrix, Inc.). AGeneChip Scanner 3000 (Affymetrix, Inc.) was used to scan the gene chip, andthe data files were obtained using GeneChip Operating Software (GCOS) (Af-

fymetrix, Inc.). Signals for all probe sets were generated by software ArrayAssist3.4 (Stratagene) using the RMA algorithm. Three biological replicates whereRNA was prepared from three independent cultures were performed for eachexperimental group (JS7131) and control group (JS71). The P value was used toassess the statistical significance of that estimate. The value ranges from 0 to 1,with a smaller P value providing more confidence in the regulation pattern. Forthis study, data with P values of �0.05 are generally considered confident andreliable. DAVID tools (http://david.abcc.ncifcrf.gov/home.jsp) were used for thefunctional group analysis. Only genes with a �2-fold change in expression levelswere submitted for this functional group analysis.

Real-time PCR. To verify the gene chip results, real-time PCR was employed byanalyzing selected genes. The primers used for each selected gene were designedusing Primer Express software v3.0 (Applied Biosystems) and synthesized by Inte-grated DNA Technologies. Reactions were set up as follows: 10 �l cDNA at 20ng/ml, 12.5 �l Power SYBR green PCR master mix (Applied Biosystems), and 1.25�l each of forward and reverse primers, each at 0.2 mM. PCR was carried out at50°C for 2 min and 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°Cfor 1 min. Samples were collected at each 60°C step using a 7300 real-time PCRmachine from Applied Biosystems and analyzed by 7300 system SDS software usinggene glgA as a reference, because it showed no change in gene chip data. For thereal-time PCR data, we include the error bars (representing the standard deviation)that were calculated from triplicates for each gene.

Protein sample preparation for 2D gel experiments. Cells were grown asdescribed above as 6-liter cultures of each strain for the protein sample prepa-ration. The protocol for preparing the E. coli inner membrane fraction wasadopted from reference 9, with some modifications. The inner membrane proteinleader peptidase and the cytosolic protein GroEL were used in a Western blotstudy to confirm that the soluble and inner membrane fractions were purified.After 5 h of growth in LB medium containing glucose, JS7131 cells becamefragile, so all steps for making spheroplasts were omitted. Cells were resus-pended in ice-cold DE buffer (1 mM dithiothreitol [DTT] and 20 mM EDTA, pH7.2) and French pressed. The French-pressed cells were spun down at 6,000 � gfor 10 min to remove the cell debris, and then the supernatant was centrifugedfor 1 h at 4°C at 250,000 � g to pellet the membranes. Supernatant was saved forthe soluble fraction protein sample. The membranes were subjected to sucrosegradient centrifugation to isolate the inner membrane vesicles (IMVs) (8). Theisolated IMVs were pelleted and were resuspended in 50 mM Tris-HCl, 1 mMMgOAc, 1 mM DTT, and 2% dodecyl maltoside (DDM). Protein concentrationwas determined using the bicinchoninic acid (BCA) kit from Pierce. All samplesused in the 2D gel experiments had a protein concentration of 10 mg/ml.

The Proteome Works system from Bio-Rad was used to perform the 2D gelexperiment. For isoelectric focusing (IEF), a mixture of 8 M urea, 2% CHAPS{3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}, and 2 mMtributyl phosphine (TBP) buffer was used for the soluble fraction and a mixtureof 7 M urea, 2 M thiourea, 2% CHAPS, 2% sulfabetaines (B3 to -10), and 2 mMTBP buffer was used for inner membrane protein fraction. Two different condi-tions were used to analyze membrane proteins: typically, pH 3 to 10 IEF stripswere used for the inner membrane fraction, but for better resolution, pH 4 to 7IEF strips were employed. Gels were stained with SYPRO ruby to achieve highsensitivity. Gel figures were obtained by the VersaDoc system and comparedusing PDquest Version 7.40 from Bio-Rad. Selected spots were cut out byProtean 2D Spotcutter and sent for in-gel digestion and mass spectrometry (MS).

In-gel digestion. The in-gel protease digestion and mass spectrometry wereperformed at the Campus Chemical Instrument Center, Ohio State University.In-gel digestion was performed using the Montage in-gel digestion kit (Millipore,Bedford, MA) following the manufacturer’s protocol. Protein spots were cut outand incubated in 50% methanol–5% acetic acid for 1 h twice and dehydrated inacetonitrile. Rehydration was done in DTT solution (5 mg/ml in 100 mM am-monium bicarbonate) for 30 min. Iodoacetamide (15 mg/ml) in 100 mM ammo-nium bicarbonate solution was then added to the rehydrated gel spots, and themixture was incubated for 30 min in the dark. After that, gel spots were washedusing acetonitrile and ammonium bicarbonate (100 mM) and dried in a SpeedVac.To digest the proteins, first, 50 �l of sequencing-grade trypsin (Promega Madi-son, WI) or chymotrypsin (Roche Indianapolis, IN) at 20 �g/ml in 50 mMammonium bicarbonate was added to the dried gel pieces for 10 min. Then, 20�l of 50 mM ammonium bicarbonate was added to the soaked gel and thedigestion was carried out overnight at room temperature. The resulting peptideswere extracted from the polyacrylamide gel with 50% acetonitrile and 5% formicacid and concentrated to a volume of about 25 �l.

Mass spectrometry. Peptide detection and sequencing were performed usinga capillary-liquid chromatography-nanospray tandem mass spectrometer (nano-LC/MS/MS). Samples were injected onto the trapping column (LC-Packings;Dionex Co., Sunnyvale, CA) and washed with 50 mM acetic acid. A ProteoPep

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II C18 column with a dimension of 5 cm and 75-�m inside diameter (NewObjective, Inc., Woburn, MA) was packed directly in the nanospray tip and usedfor chromatographic separations. Peptides were eluted off the column into theLTQ system (Thermo Finnigan) using a gradient of 2 to 80% acetonitrile over 50min, with a flow rate of 300 nl/min. The MS/MS was performed under standardconditions. Briefly, a nanospray source was operated in positive-ion mode with a3-kV spray voltage and with a 200°C capillary temperature. A full scan wasrecorded first between 350 and 2,000 Da, followed by another MS/MS scan togenerate product ion spectra to determine the amino acid sequence of the 10most abundant peaks in the spectrum. The collision-induced dissociation (CID)fragmentation energy was set to 35%. Dynamic exclusion was enabled with arepeat count of 30 s, exclusion duration of 350 s, a low-mass width of 0.5 Da, anda high-mass width of 1.50 Da.

The raw data from the MS/MS experiment containing sequence informationwere analyzed by Mascot Daemon by Matrix Science version 2.2.1 (Boston, MA)and matched against SwissProt database version 54.1. During analysis, methio-nine oxidation and carbamidomethyl cysteine modifications were considered;two missed cleavages from the enzyme digestion were permitted. Only proteinswith a Mascot score of 50 or higher, a P value of �0.05, and at least two uniquematched peptides were considered as positive hits. The identified proteins werethen individually checked manually by matching their calculated molecular mass,pI, and predicted cellular localization data with the 2D gel data to give the finalassignment.

PspA induction and accumulation of pro-OmpA in carbonyl cyanide m-chlo-rophenylhydrazone (CCCP)-treated cells. To determine the concentration ofCCCP required for pro-OmpA to accumulate in the cytoplasm of the cell, thecultures were treated with different concentrations of CCCP. Specifically, JS7131cells were grown in LB medium plus 0.2% arabinose for 5 h and then transferredinto M9 medium containing 0.5% fructose for 15 min and treated with various

concentrations of CCCP (dissolved in DMSO) for 15 min prior to labeling with[35S] methionine for 1 min. For the control group, equal amounts of DMSO wereadded to the LB medium before radiolabeling the cells. Also, JS7131 was grownin LB medium with glucose to deplete YidC and analyzed the same way, exceptno CCCP was added. Samples were analyzed by immunoprecipitation withOmpA antibody (from our lab collection). To measure the effects of CCCP onthe growth of JS7131 in LB medium plus 0.2% arabinose, JS7131 cells weregrown in LB medium for 2 h to the mid-log phase. CCCP was then added to theculture at different concentrations, and OD600 was measured for an additional3 h. We also grew JS7131 in LB medium containing 0.2% glucose or 0.2%arabinose with no CCCP treatment. For the PspA induction study, JS7131 cellswere treated with CCCP the same way as for the growth curve analysis. SDS-PAGE was performed using a 12% polyacrylamide gel. Where indicated, the gelwas stained with Coomassie brilliant blue G-250 or analyzed by Western blotting.To reduce the amount of background bands on the Coomassie-stained gel, thesoluble proteins in the samples were extracted from the E. coli cells using theB-PER bacterial protein extraction reagent (Pierce).

Western blotting. The proteins YidC, PspA, maltose binding protein (MBP),DnaJ, and GroEL were analyzed to confirm the protein levels as determined by2D gel analysis. They were detected by immunoblot analysis using the SuperSignalWest Pico chemiluminescent substrate (Pierce). The antibodies used were raisedagainst YidC (from our lab), PspA (Jan Tommassen at Utrecht University),MBP (New England Biolabs), DnaJ (Stressgen Biotechnologies), and GroEL(Sigma). Anti-DnaJ, anti-GroEL, and anti-YidC are polyclonal antibodies gen-erated in rabbits against purified proteins or peptides. Anti-MBP is a monoclonalantibody (murine). As the control for the inner membrane proteome, Foa, Fob,Foc, and MalF were also analyzed by Western blotting under the YidC depletioncondition. Antibodies against the Foa, Fob, and Foc subunits of F1Fo ATPasewere kindly provided by Robert H. Fillingame at the University of Wisconsin—Madison. MalF antibody was provided by Beth Traxler at the University ofWashington. Inner membrane proteins were purified using sucrose gradients andultracentrifugation following a previously described protocol (9).

RESULTS

YidC depletion affects growth and cell morphology. Thegrowth of JS71 and JS7131 was measured at 37°C after 0.2%glucose was added to the medium. Since YidC synthesis wasrepressed in JS7131, its growth rate slowed down significantlycompared to that of JS71 (Fig. 1), and after 7 h, JS7131stopped growing. We confirmed by Western blotting that YidCis depleted in JS7131 to �90% after 5 h of growth in LBglucose medium (see Fig. S1 in the supplemental material). Itshould be noted that JS71 is entering the stationary phase atthe 4-h time point (for the semi-log plot, see Fig. S2 in thesupplemental material).

The cell morphology was examined using a microscope un-der YidC expression and YidC depletion conditions. Figure 2reveals the YidC-depleted JS7131 cells grown for 5 h withglucose were significantly longer than JS71 cells (which showed

FIG. 1. YidC depletion affects cell growth in liquid medium.Growth of JS71 (control) and JS7131 (YidC depletion) was measuredby OD600. Absorbances were measured from 3 independent cultures ofboth JS71 and JS7131. A 0.2% concentration of glucose was added attime point 0. Error bars represent standard deviation from the 3independent culture measurements.

FIG. 2. Cell morphology is affected by YidC depletion. Images (�100 magnification) were obtained after the cells were grown for 5 h underthe YidC depletion condition for JS7131 (right panel) or the normal condition for JS71 (left panel).

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normal E. coli morphology) and were nonmotile. The resultsindicate that the cells did not divide normally when YidC wasdepleted, possibly due to a disruption of the membrane struc-ture.

Transcriptome analysis during the YidC depletion condi-tion. (i) Overall regulation pattern. To better understand thechanges in gene expression during YidC depletion, a high-throughput experiment was carried out under glucose condi-tions using JS71 grown as a control sample and JS7131 as a testsample. Gene chip techniques were employed to comprehen-sively analyze the change of the transcriptome pattern duringthe YidC depletion condition compared to the normal condi-tion. According to the gene chip results (see the Excel fileshowing the entire data set in the supplemental material),among the total of 10,208 probes (which represent 4,355 genesand 1,427 intergenic regions) on the gene chip, 449 probesshowed �2-fold upregulation, while 317 probes showed �0.5-fold downregulation, both with P values of �0.05. The upregu-lated probes include 163 genes, 129 hypothetical genes orundefined transcripts, and 157 intergenic regions. The down-regulated probes include 99 genes, 173 hypothetical genes orundefined transcripts, and 45 intergenic regions. Table 1 liststhe results with �3-fold changes, and Table 2 lists those for�0.3-fold changes, each with a P value of �0.05. Some geneswhich showed significant change in their expression levels withrelatively high copy numbers were selected for real-time PCRexperiments to confirm the gene chip data (Fig. 3). Amongothers, this included the strongly upregulated cadA and pspAgenes and the strongly downregulated flu gene. We also exam-ined the expression of these genes at 3 h as well. Real-timePCR confirmed that the mgtA, cadA, tig, and pspA genes werestrongly induced at 5 h of YidC depletion as well as at the 3-hYidC depletion time. It also confirmed that the expression ofYidC which is controlled by the araBAD promoter was re-pressed by growth of JS7131 in LB-glucose medium. As ex-pected, real-time PCR showed expression of flu was repressed.

Next, we used DAVID tools to group the significantly af-fected genes (larger than 2-fold and less than 0.5-fold) by theirfunctions. To make the analysis easier, only genes with aknown function were submitted to the program. The resultsfrom this grouping analysis are summarized in Fig. 4.

(ii) Stress response. Since YidC is an inner membrane in-sertase and chaperone, depletion of YidC will affect the inser-tion and folding of its substrates, thus leaving these proteinseither in the incorrect cellular location or in the incorrectfolding state. At the same time, some of YidC’s substrates,such as F1Fo ATP synthase subunits a and c and CyoA (subunitII of cytochrome bo oxidase) have important physiologicalfunctions in energy transduction for the cell when assembledinto their respiratory complex (39). Therefore, it is conceivablethat depletion of YidC would trigger a stress response system,allowing the cell to handle potential problems, which includeboth primary effects from the absence of YidC and secondaryeffects from the absence of YidC’s substrates. Indeed, wefound that the phage shock response, cold shock response, andthe Cpx pathway were switched on following YidC depletion(Table 1).

Both the phage shock protein family and the cold shockprotein family genes were highly upregulated (Table 1). pspA,-B, -C, -D, and -E showed 13.6-, 8.3-, 6.8-, 8.0-, and 3.4-fold

upregulation, respectively. For the cold shock protein family, 5out of 9 cold shock proteins were increased at the mRNA level,including cspA, -B, -C, -G, and -I (Table 1; see the Excel file inthe supplemental material). By microarray analysis, cspA is thegene with the highest upregulation, at 20-fold. Previous studiesshowed that cold shock proteins are generally mRNA-bindingproteins which handle the secondary structure of mRNA,working together with some RNA helicases. Interestingly, twoRNA helicase genes, deaD and rhlE (see the Excel file in thesupplemental material) were also upregulated in our YidCdepletion experiments.

Previous reports have shown that Cpx/sigma E pathwayswere switched on during the YidC depletion condition (32). Inour data, 3 isomerase genes from the Cpx pathway, fkpA (1.55-fold) (coding for an FK506 binding protein [FKBP]-type pep-tidyl-prolyl cis-trans isomerase), dsbA (1.59-fold) (a periplas-mic protein disulfide isomerase), and ppiA (3.48-fold) (codingfor peptidyl-prolyl cis-trans isomerase A) were upregulated(see the Excel file in the supplemental material). Unfortu-nately, the cpxP probe is missing from our gene chip probearray. Therefore, we set up an additional real-time PCR ex-periment to check the cpx transcription profile. The resultshowed a 15-fold upregulation of cpxP at 5 h YidC depletion(Fig. 3). In contrast, rpoH, which was used as the reporter foractivation of the sigma E pathway (32), showed no significantchange (see the Excel file in the supplemental material). Tofurther investigate the activation of both pathways, 5 genesfrom the Cpx pathway and 6 genes from the sigma E pathwaywere selected and their expression profiles under the YidCdepletion condition were examined by real-time PCR (Fig. 5).For the Cpx pathway, all 3 genes (dsbA, ompC, and ppiA) thatare expected to be upregulated if the pathway is switched onwere upregulated at both 3 and 5 h, while motB and flgM,which are expected to be downregulated if the pathway isswitched on, were downregulated at both time points (Fig. 5).In contrast, at the 3-h time point, only half of the selectedgenes (ropE, mdoG, and ompA) in the sigma E pathwaymatched their expected regulation patterns if the pathwaywas switched on. Others were regulated in the oppositemanner (cutC and ompX) (Fig. 5). At the 5-h time point,none of the sigma E genes showed significant changes inexpression when YidC was depleted. degP, which typically isupregulated when either the sigma E or Cpx stress pathwayis activated, was not significantly changed (Fig. 5; see theExcel file in the supplemental material).

We conclude that the Cpx pathway is switched on basedon the fact that CpxP is induced and the fact that a numberof genes in this pathway (see Table S1 in the supplementalmaterial) are switched on upon YidC depletion. Increasedinduction of CpxP has been shown to be a good indicatorthat the Cpx pathway is activated (5); i.e., CpxP can only beswitched on by activation by this stress pathway (T. Silhavy,personal communication). We suspect that the Cpx path-way-controlled genes that were not induced in our microar-ray study (see Table S1 in the supplemental material) wereinfluenced by other factors that occurred during YidC de-pletion. Many of these genes have other promoters in theregulon and show complex regulation (7). The evidence thatthe sigma E pathway is activated in our microarray study isnot strong (see Discussion) because there are a number of



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TABLE 1. Significantly upregulated genes in this studya

Probe identificationFold change inexpression inJS7131/JS71

Pvalue Gene name Functional annotationb

DNA/RNA related1768419_s_at 3.22 5E�03 exo 5�33� exonuclease1766724_s_at 3.49 1E�02 recD DNA helicase, ATP-dependent dsDNA/ssDNA exonuclease V subunit, ssDNA endonuclease1766069_s_at 3.25 5E�04 deaD Inducible ATP-independent RNA helicase1768185_at 3.97 4E�02 ybfD Putative DNA ligase1761660_s_at 3.59 1E�02 rne RNase E1760225_s_at 3.16 5E�02 rpoB RNA polymerase, �-subunit1767680_at 3.16 7E�03 Regulatory RNA

Energy1759374_s_at 3.34 3E�02 cyoB Cytochrome o ubiquinol oxidase subunit I1769018_s_at 6.70 1E�03 cyoA Cytochrome o ubiquinol oxidase subunit II1764404_s_at 4.31 3E�02 cyoD Cytochrome o ubiquinol oxidase subunit IV1761759_s_at 3.79 5E�02 nuoB NADH dehydrogenase I chain B1763447_s_at 4.87 2E�02 nuoM NADH dehydrogenase I chain M1761548_s_at 3.15 3E�02 bax Putative ATP-binding protein1767503_s_at 3.50 2E�02 yihK Putative GTP-binding factor1768459_s_at 3.38 2E�02 yhjL Putative oxidoreductase subunit1764381_s_at 3.30 5E�02 atpF Membrane-bound ATP synthase, Fo sector, subunit b

Fimbrin1767098_s_at 4.05 1E�02 fimA Major type 1 subunit fimbrin (pilin)1763102_s_at 3.16 3E�03 fimI Fimbrin-like protein FimI precursor

Metabolism1769070_s_at 3.49 2E�04 dxs 1-Deoxyxylulose-5-phosphate synthase; flavoprotein1767819_s_at 3.02 9E�03 accB Acetyl-CoA carboxylase, BCCP subunit; carrier of biotin1762548_s_at 5.46 4E�05 apt Adenine phosphoribosyltransferase1761486_s_at 3.55 9E�04 add Adenosine deaminase1762363_at 4.84 3E�02 malS �-Amylase1759122_s_at 3.03 2E�05 thrA Aspartokinase I, homoserine dehydrogenase I1761305_s_at 3.30 2E�03 mrdA Cell elongation, e phase; peptidoglycan synthetase; penicillin-binding protein 21762501_s_at 3.56 5E�02 fdoH Formate dehydrogenase O, iron-sulfur subunit1762677_s_at 3.18 9E�03 gatA Galactitol-specific enzyme IIA of PTS system1762781_at 3.59 5E�02 aceK Isocitrate dehydrogenase kinase/phosphatase1764617_s_at 32.86 1E�03 cadA Lysine decarboxylase 11769223_s_at 3.07 6E�03 argA N-Acetylglutamate synthase; amino acid acetyltransferase1764499_s_at 3.17 1E�02 pldA Outer membrane phospholipase A1760363_s_at 5.12 2E�03 ppsA Phosphoenolpyruvate synthase1769056_s_at 3.80 1E�02 hemK Possible protoporphyrinogen oxidase1766494_s_at 4.31 4E�07 yiaE Putative dehydrogenase1764702_s_at 5.73 4E�02 yhdG Putative dehydrogenase1763775_at 6.69 5E�03 Putative glycan biosynthesis enzyme1767386_at 8.77 3E�02 Putative ligase1762947_s_at 11.68 1E�02 gatZ Putative tagatose 6-phosphate kinase 11768388_s_at 5.07 4E�03 glpD sn-Glycerol-3-phosphate dehydrogenase (aerobic)1763273_s_at 6.15 9E�04 sodA Superoxide dismutase, manganese1767672_s_at 8.40 7E�03 gatY Tagatose-bisphosphate aldolase 11763484_s_at 6.92 5E�02 thiC Thiamine biosynthesis, pyrimidine moiety1764403_s_at 4.31 4E�03 thiE Thiamine biosynthesis, thiazole moiety1767599_s_at 3.71 9E�03 thyA Thymidylate synthetase1764322_at 3.81 3E�02 avtA Valine-pyruvate aminotransferase1767715_s_at 3.62 2E�04 Putative polymerase proteinase1763494_s_at 3.74 1E�03 Putative amidase1763111_s_at 3.41 4E�02 Acetolactate synthase II, valine insensitive, large subunit, silent in K-121766804_s_at 3.81 1E�02 glpK Glycerol kinase

Transporter1765773_s_at 3.13 5E�03 oppC Homolog of Salmonella oligopeptide transport permease protein1768775_s_at 5.57 5E�03 rbsB D-Ribose periplasmic binding protein1760971_s_at 5.32 1E�03 rbsA ATP-binding component of D-ribose high-affinity transport system1760798_s_at 3.20 2E�03 proV ATP-binding component of transport system for glycine, betaine, and proline1760041_s_at 3.26 1E�03 malK ATP-binding component of transport system for maltose1759395_s_at 3.88 9E�03 dppA Dipeptide transport protein1763403_s_at 4.78 1E�04 rbsC D-Ribose high-affinity transport system1763733_s_at 7.82 5E�02 rbsD D-Ribose high-affinity transport system, membrane-associated protein1764804_s_at 3.77 3E�03 pstS High-affinity phosphate-specific transport system, periplasmic phosphate-binding protein1764279_s_at 13.33 1E�02 mgtA Mg2 transport ATPase, P-type 11766260_s_at 3.67 2E�03 tsx Nucleoside channel, receptor of phage T6 and colicin K1768919_s_at 3.95 7E�04 sdaC Probable serine transporter1768684_s_at 3.78 1E�02 manX PTS enzyme IIAB, mannose specific1763069_s_at 3.74 3E�02 manY PTS enzyme IIC, mannose specific1762917_s_at 7.26 2E�05 ptsG PTS system, glucose-specific IICB component1764801_at 3.28 5E�02 yieG Putative membrane/transport protein1765980_s_at 6.93 5E�03 yjdL Putative peptide transporter1764271_s_at 3.04 6E�03 yhiV Putative transport system permease protein1760764_s_at 6.31 4E�05 Putative transporter protein1759972_s_at 20.69 1E�02 cadB Transport of lysine/cadaverine

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genes that are regulated in the opposite direction, and somegenes that are known to be highly expressed by activation ofthe sigma E pathway were not upregulated (see Table S2 inthe supplemental material).

(iii) Proteases and chaperones. Proteases and chaperonesare expected to play important roles in handling malfoldedmembrane proteins during the YidC depletion condition. Thegenes coding for the two chaperones, fimC (periplasmic chap-

TABLE 1—Continued

Probe identificationFold change inexpression inJS7131/JS71


Protease and chaperone1763873_s_at 3.48 8E�06 ppiA Peptidyl-prolyl cis-trans isomerase A (rotamase A)1762546_s_at 4.49 2E�02 fimC Periplasmic chaperone, required for type 1 fimbriae1764696_s_at 3.74 4E�05 ptr Protease III1767122_s_at 4.85 4E�03 tig Trigger factor, a molecular chaperone involved in cell division

Stress response1761114_s_at 20.03 3E�04 cspA Cold shock protein 7.4, transcriptional activator of hns1762932_s_at 4.56 6E�03 cspB Cold shock protein; may affect transcription1765796_s_at 8.00 2E�05 pspD Phage shock protein1768435_s_at 8.30 2E�04 pspB Phage shock protein1760618_s_at 3.38 5E�04 pspE Phage shock protein1769101_s_at 13.60 5E�04 pspA Phage shock protein, inner membrane protein1762530_s_at 6.78 8E�03 pspC Phage shock protein, activates phage shock protein expression1769169_s_at 3.12 2E�02 yjiY Putative carbon starvation protein

Translation and transcription1762512_s_at 4.10 2E�03 rpsA 30S ribosomal subunit protein S11767291_s_at 3.32 4E�02 rpsJ 30S ribosomal subunit protein S101765175_s_at 3.48 4E�03 rpsP 30S ribosomal subunit protein S161768873_s_at 3.19 5E�02 rpsB 30S ribosomal subunit protein S21762255_s_at 3.08 2E�02 rplR 50S ribosomal subunit protein L181767377_s_at 3.62 2E�02 rplS 50S ribosomal subunit protein L191760752_s_at 3.44 5E�02 rplB 50S ribosomal subunit protein L21761244_s_at 3.25 3E�02 rplV 50S ribosomal subunit protein L221761605_s_at 3.47 2E�03 glyQ Glycine tRNA synthetase, �-subunit1765772_s_at 3.54 5E�03 glyS Glycine tRNA synthetase, �-subunit1761999_s_at 4.08 4E�02 fusA GTP-binding protein chain elongation factor EF-G1767618_s_at 4.06 9E�04 prfA Peptide chain release factor RF-11766499_s_at 3.09 1E�03 pheS Phenylalanine tRNA synthetase, �-subunit1764306_s_at 3.32 4E�05 rnpA RNase P, protein component; protein C5; processes tRNA, 4.5S RNA1765454_s_at 3.24 7E�04 rho Transcription termination factor Rho, polarity suppressor1764493_s_at 3.46 4E�03 trmD tRNA methyltransferase; tRNA (guanine-7-)-methyltransferase1759109_s_at 3.01 1E�02 Putative elongation factor

Other1762757_s_at 3.18 4E�02 pal Peptidoglycan-associated lipoprotein1767981_s_at 3.66 4E�03 ompF Outer membrane protein 1a (Ia, b, F)1766425_s_at 4.11 4E�02 priB Primosomal replication protein N1761338_s_at 5.59 1E�03 Putative Bor protein of prophage CP-933X1765995_s_at 3.65 6E�03 Putative membrane protein

Hypothetical1760914_s_at 7.69 3E�04 Hypothetical protein1766415_s_at 6.88 5E�04 Hypothetical protein1764179_s_at 3.64 8E�04 Hypothetical protein1761529_s_at 3.41 2E�03 Hypothetical protein1759951_s_at 3.55 2E�02 Hypothetical protein1766884_s_at 3.60 4E�02 Hypothetical protein1762024_s_at 3.31 1E�02 Hypothetical protein1767540_s_at 5.41 1E�02 yeaJ ORF, unknown function1769192_s_at 5.41 5E�06 yeaF ORF, hypothetical protein1763019_s_at 4.73 4E�04 yafK ORF, hypothetical protein1767568_s_at 3.25 8E�04 yhjU ORF, hypothetical protein1762187_s_at 3.30 3E�03 ORF, hypothetical protein1760906_s_at 4.87 5E�03 ORF, hypothetical protein1766652_s_at 4.14 5E�03 ORF, hypothetical protein1762056_s_at 3.15 6E�03 ynaE ORF, hypothetical protein1760762_s_at 4.06 7E�03 ORF, hypothetical protein1766638_s_at 3.84 9E�03 ybeA ORF, hypothetical protein1763486_s_at 3.14 1E�02 yceD ORF, hypothetical protein1759527_s_at 3.01 1E�02 yhjW ORF, hypothetical protein1761324_at 4.36 2E�02 ORF, hypothetical protein1764771_s_at 4.36 2E�02 ycbK ORF, hypothetical protein1766279_at 8.09 3E�02 ycgW ORF, hypothetical protein1767883_s_at 4.82 3E�02 yfjA ORF, hypothetical protein1762710_s_at 3.43 4E�02 ybgF ORF, hypothetical protein1765691_s_at 3.13 5E�02 ORF, hypothetical protein

a The most significantly upregulated genes from the gene chip data are shown. Only those showing a �3-fold change with reliable P values (P � 0.05) are listed.b dsDNA, double-stranded DNA; ssDNA, single-stranded DNA; acetyl-CoA, acetyl coenzyme A; BCCP, biotin carboxyl carrier protein; PTS, phosphotransferase;

ORF, open reading frame.



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TABLE 2. Significantly downregulated genes in this studya

Protein type and probeidentification

Fold change inexpression inJS7131/JS71


Energy1767636_s_at 0.22 1E�03 frdA Fumarate reductase, anaerobic, flavoprotein subunit1767792_s_at 0.24 3E�03 appC Probable third cytochrome oxidase, subunit I1767833_s_at 0.23 2E�04 appB Probable third cytochrome oxidase, subunit II1760548_s_at 0.27 2E�04 yhaH Putative cytochrome1759127_s_at 0.12 9E�06 yghA Putative oxidoreductase1764339_s_at 0.24 1E�04 ybdR Putative oxidoreductase

Metabolism1767109_s_at 0.28 3E�04 hdhA 7-�-Hydroxysteroid dehydrogenase1768184_s_at 0.23 7E�06 hyaB Hydrogenase-1, large subunit1765453_s_at 0.19 8E�04 hyaA Hydrogenase-1, small subunit1768953_s_at 0.26 1E�04 appA Phosphoanhydride phosphorylase; pH 2.5 acid phosphatase; periplasmic1768393_s_at 0.23 9E�05 purE Phosphoribosylaminoimidazole (AIR) carboxylase, catalytic subunit1762205_s_at 0.20 2E�04 purC Phosphoribosylaminoimidazole-succinocarboxamide (SAICAR) synthetase

Transporter1768513_x_at 0.19 3E�02 KdpF protein of high-affinity potassium transport system1760130_s_at 0.25 2E�05 yohK Putative seritonin transporter

Stress response1763125_s_at 0.09 7E�05 glgS Glycogen biosynthesis, rpoS dependent1761076_s_at 0.25 1E�04 psiF Induced by phosphate starvation1759179_s_at 0.14 2E�04 osmB Osmotically inducible lipoprotein

Translation and transcription1762381_s_at 0.27 3E�03 Spot 42 RNA1763089_s_at 0.29 8E�04 6S RNA1763985_s_at 0.25 6E�03 4.5S RNA, component of ribonucleoprotein particle1765371_s_at 0.18 1E�05 osmE Activator of ntrL gene

Other1760228_s_at 0.23 2E�05 Putative filament protein1761941_s_at 0.30 4E�05 Putative polyprotein1761735_s_at 0.30 2E�05 Amino-terminal fragment of WrbA1766745_s_at 0.28 1E�04 Entericidin B1760040_at 0.08 7E�04 flu Outer membrane fluffing protein, similar to adhesin1763857_s_at 0.17 4E�04 relF Polypeptide destructive to membrane potential1763864_s_at 0.20 2E�04 Putative homeobox protein1767642_s_at 0.11 3E�02 yibP Putative membrane protein1762401_s_at 0.22 2E�04 yciF Putative structural proteins1768436_s_at 0.07 4E�031760679_at 0.17 1E�03

Hypothetical1761754_s_at 0.08 1E�04 Hypothetical protein1759995_s_at 0.25 1E�02 Hypothetical protein1761327_s_at 0.27 2E�03 Hypothetical protein1767107_s_at 0.28 3E�02 Hypothetical protein1763445_s_at 0.07 5E�04 Hypothetical protein1760150_s_at 0.30 4E�02 Hypothetical protein1760953_at 0.18 2E�02 ORF, unknown function1765333_s_at 0.21 2E�02 ORF, unknown function1760940_s_at 0.29 6E�04 ORF, unknown function1766731_s_at 0.09 5E�05 yohJ ORF, hypothetical protein1761873_s_at 0.09 8E�05 yciG ORF, hypothetical protein1759494_s_at 0.13 9E�05 ycfR ORF, hypothetical protein1768326_s_at 0.14 2E�04 ydeI ORF, hypothetical protein1766187_s_at 0.15 6E�06 ORF, hypothetical protein1763516_s_at 0.19 3E�04 yceP ORF, hypothetical protein1760727_s_at 0.19 3E�05 ygaM ORF, hypothetical protein1766489_s_at 0.19 2E�06 ybfA ORF, hypothetical protein1762657_s_at 0.20 1E�03 ORF, hypothetical protein1764792_s_at 0.21 4E�04 yhbO ORF, hypothetical protein1761638_s_at 0.22 3E�03 ymgE ORF, hypothetical protein1761022_s_at 0.23 3E�03 yecH ORF, hypothetical protein1764073_s_at 0.23 2E�03 yhcO ORF, hypothetical protein1762735_s_at 0.24 9E�04 yhhA ORF, hypothetical protein1768189_s_at 0.24 8E�04 yphA ORF, hypothetical protein1768649_at 0.25 1E�05 ycdF ORF, hypothetical protein1768587_s_at 0.26 4E�04 yciE ORF, hypothetical protein1761690_s_at 0.26 1E�03 ORF, hypothetical protein1767977_s_at 0.26 4E�04 yjfY ORF, hypothetical protein1766480_s_at 0.27 1E�03 ORF, hypothetical protein1768363_s_at 0.27 3E�04 ybdQ ORF, hypothetical protein1766893_s_at 0.28 6E�05 ORF, hypothetical protein1764881_s_at 0.28 2E�04 ybeH ORF, hypothetical protein1765823_s_at 0.29 2E�04 yeaQ ORF, hypothetical protein

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erone) and tig (trigger factor, a chaperone involved in proteinfolding), were highly induced at 4.5- and 4.9-fold, respectively(Table 1). Surprisingly, other chaperone genes, namely, dnaK,dnaJ, groES, and groEL, were not upregulated in our microar-ray analysis (see the Excel file in the supplemental material), incontrast to a recent microarray study of Streptococcus mutanswhen the signal recognition particle (SRP) component Ffh wasknocked out (10).

Several protease genes were shown to be significantly up-regulated upon YidC depletion (greater than 2-fold change)(see the Excel data set in the supplemental material), includingompT, ptr (protease III), hflC, and hflK, which form a mem-brane complex with the membrane protease hflB (FtsH) andnegatively regulate the protease activity of FtsH. Some other

protease genes such as iap (aminopeptidase in alkaline phos-phatase), yaeL, lspA, htpX, glpG, and hflB were also induced(140% to 192% compared to the control).

(iv) The role of phage shock response in YidC-depleted cells.The phage shock response is thought to help maintain theproton motive force (PMF) under membrane stress conditionssuch as infection by phage or treatment with a protonophore(13). Since YidC depletion results in a decrease in the PMFdue to a defect in the assembly and activity of cytochrome bo3

oxidase and F1Fo ATP synthase (39), we expected that thephage shock response is induced after YidC depletion. There-fore, we compared PspA expression after YidC depletion withthe effects of addition of CCCP, a protonophore to collapse thePMF, to E. coli cells (22).

Surprisingly, our microarray data derived from YidC-de-pleted cells are very different from the data from another genechip study in which the effects of CCCP were analyzed (22).That study did not see an activation of the Psp operon whenCCCP was added, although transcription of a number of othergenes was affected (see Table S3). We found significant PspAinduction upon YidC depletion (Fig. 6A). Equal amounts(with cells normalized to the same OD600) of JS7131 cellsgrown in glucose (YidC-depleted conditions), arabinose (YidCexpression conditions), and arabinose containing various con-centrations of CCCP were collected and subjected to SDS-PAGE and Western blot analysis (Fig. 6A). When YidC wasdepleted, PspA was induced (see arrowhead, Glc, upperpanel), while no PspA was detectable on the Coomassie-stained gel after CCCP treatment (Fig. 6A, upper panel). Sim-ilarly, Western blot analysis using PspA antibody showed thatthere is a strongly induced PspA band in YidC-depleted cellsand only a weak PspA band in CCCP-treated cells.

The effects of the different concentrations of CCCP on thegrowth of the cell were analyzed. We found that the addition ofCCCP (see arrow) at concentrations above 2.5 �M slowedgrowth of JS7131 (in LB medium supplemented with 0.2%arabinose). The growth curves of the cultures treated with 5and 12.5 �M CCCP were comparable to that of YidC-depletedJS7131 cells (Fig. 6B and C). After the addition of 25 �MCCCP, the cells stopped growing. The effects of CCCP onpro-OmpA export were examined. Pro-OmpA only accumu-lated when CCCP was added at 40 �M (Fig. 6D). Our effectsof CCCP on pro-OmpA accumulation are similar to what Pos-sot et al. (23) had reported: i.e., 50 �M CCCP caused analmost complete block in pullulanase secretion. They showed

TABLE 2—Continued

Protein type and probeidentification

Fold change inexpression inJS7131/JS71


1765253_s_at 0.29 7E�05 yjbJ ORF, hypothetical protein1767891_s_at 0.29 2E�04 ynhG ORF, hypothetical protein1767800_s_at 0.30 2E�04 yqjI ORF, hypothetical protein1768822_s_at 0.30 8E�04 ybiM ORF, hypothetical protein1759909_s_at 0.30 1E�05 ORF, hypothetical protein1768573_s_at 0.30 8E�04 ORF, hypothetical protein1761089_at 0.30 3E�04 relE ORF, hypothetical protein1768030_s_at 0.30 3E�05 ytfK ORF, hypothetical protein1766739_s_at 0.30 8E�04 ychH ORF, hypothetical protein

a The most significantly downregulated genes from the gene chip data are shown. Only those with �0.3-fold changes with reliable P values (P �0.05) are listed.b ORF, open reading frame.

FIG. 3. Selected genes are tested by real-time PCR to confirm themicroarray data. The y axis represents the relative expression level ofeach gene under YidC depletion conditions compared to the normalcondition. A value of 1 would represent no change in expression uponYidC depletion. The mRNA levels at both the 3- and 5-h time pointswere tested. Error bars are calculated from triplicates for each exper-iment.



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that already 5 �M CCCP dissipated the �� below the sensi-tivity of the assay using tetraphenyl phosphonium ion (TPP)uptake, suggesting the PMF was almost completely collapsed.In conclusion, our PspA study here shows that the PspA in-duction that is observed upon YidC depletion appears inde-pendent of the PMF.

(v) Other possible effects. Another reason why the expres-sion of some genes show changes in our study may be due toJS71, but not JS7131, entering stationary phase after 5 h ofgrowth (Fig. 1; see Fig. S1 in the supplemental material). Forexample, stationary-phase-induced genes would be switched onin JS71 because it is entering stationary phase at the 4-h point.Since JS7131 is not entering stationary phase, this could lead toa decrease in the expression of those genes in JS7131 com-pared to JS71.

To evaluate whether this is influencing our microarray data,we performed real-time PCR at 3 and 5 h on two genes that areknown to be induced at stationary phase. The stationary-phase-induced genes aidB and bolA were selected becausetranscription of these genes is markedly increased upon enter-ing stationary phase (34) (see Table S4 in the supplementalmaterial). The aidB gene encodes a dehydrogenase that has arole in DNA metabolism (40); the bolA-encoded protein is a

regulator of cell wall biosynthetic enzymes (30). We comparedlevels of gene expression of aidB and bolA in JS7131 (com-pared to JS71) after 3 and 5 h of growth in LB medium plusglucose. We found that levels of gene expression of aidB andbolA were decreased at 5 h relative to the level at 3 h, consis-tent with JS71 entering stationary phase and its influence onthe gene expression data (Fig. 3). A second approach to checkif the microarray data are being influenced by the differentgrowth phases of the JS71 and JS7131 strains is to look at othergenes under rpoS control. RpoS is essential for induction ofvarious stationary-phase genes, including aidB. If these RpoS-controlled stationary-phase genes are being switched on inJS71 as it begins to enter stationary phase, it will result in thegenes being downregulated in JS7131 compared to JS71. Wesee this for the osmotically regulated gene osmB (0.14-fold) fortrehalose breakdown, the glycogen synthesis gene glgS (0.09-fold), the acid phosphatase gene appA (0.26-fold), and thebolA gene (0.40-fold) (see the Excel file in the supplementalmaterial) involved in cell morphology (Table 2). Finally, com-parison of our YidC depletion microarray data with data froma microarray study that identified 53 stationary-phase-inducedgenes (34) showed that one-third of the stationary-phase-in-duced genes were affected in our YidC depletion study (lead-

FIG. 4. Gene category summary. All genes showing a different expression pattern (�2 fold difference and P value of �0.05) were groupedaccording to their functions. The x axis indicates the number of genes within each functional group. Black bars represent those groups of geneswhich were upregulated by microarray analysis under YidC depletion conditions for 5 h. Gray bars represent those groups of genes which weredownregulated by microarray analysis under YidC depletion conditions for 5 h.



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ing to a decrease in the expression of the genes in JS7131/JS71)(see Table S4 in the supplemental material). Nevertheless, wethink that the different growth phases of JS7131 and JS71 aremarginally influencing the expression pattern of the sigma Epathway and Cpx pathway genes compared to the influence ofYidC depletion on the Cpx pathway genes (Fig. 5).

FIG. 5. Regulation of specific Cpx- and sigma E-controlled genesby real-time PCR under YidC depletion conditions. The expectedregulation pattern of genes is indicated for each pathway listed in thechart. The y axis represents the relative expression level by real-timePCR of each gene under YidC depletion conditions compared to

the normal condition. (A) Relative expression level of selected genesfor Cpx pathway at both 3 and 5 h of YidC depletion. (B) Relativeexpression level of selected genes for sigma E pathway at both 3 and5 h of YidC depletion.

FIG. 6. YidC depletion induces a higher level of phage shock re-sponse than CCCP treatment. (A) JS7131 was grown in glucose (0.2%)or arabinose (0.2%), treated with various concentrations of CCCP, andthen analyzed by Coomassie G-250 staining (top panel) or Westernblotting using PspA antibody (lower panel). Soluble proteins wereremoved before running the gel for Coomassie stain (see Materials andMethods). (B) The growth curve was measured for JS7131 in LBmedium containing arabinose (0.2%) with various concentrations ofCCCP. (C) The growth curve was determined for JS7131 in LB me-dium with arabinose (0.2%) or glucose (0.2%). (D) The addition of 40�M CCCP to the JS71 culture resulted in the accumulation of 35S-labeled pro-OmpA in the cell. The amounts of precursor and matureOmpA in a 1-min pulse-label were determined as described in Mate-rials and Methods.



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(vi) Other significantly up/downregulated genes. Table 1(and see the Excel file in the supplemental material) shows thatthe ribosome and aminoacyl-tRNA biosynthesis pathways wereaffected under YidC depletion conditions. Nineteen genes forribosomal proteins of the 30S subunit, 20 genes for ribosomalproteins of the 50S subunit, 8 tRNA synthetases, 2 elongationfactors (EFs), and 1 initiation factor (IF) were highly induced.The translocase subunit secY also showed a 2.3-fold increase atmRNA level, while neither secE nor secG was affected. Also, alarge number of genes involved in ubiquinone biosynthesis,glycine, serine, and threonine metabolism, and the ABC trans-porter system were upregulated (Table 1; see the Excel file inthe supplemental material). Interestingly, many of these trans-porter systems were upregulated in their ATP-binding compo-nents, such as rbsA (ATP-binding component of a D-ribosetransport system), pstB (ATP-binding component of a phos-phate transport system), potA (ATP-binding component ofspermidine/putrescine transport), proV (ATP-binding compo-nent of transport system for glycine, betaine, and proline), andmalK (ATP-binding component of maltose transport system)(see the Excel file in the supplemental material for potA; seeTable 1 for the other genes). The oxidative phosphorylationpathway was also affected, with NADH dehydrogenase I (1.7-to 4.9-fold upregulation; see the Excel file in the supplemen-tal material for the full list) and cytochrome bo3 oxidase(2.5- to 6.8-fold upregulation; see the Excel file in the sup-plemental material) showing upregulation for most of theircomponents at the mRNA level, while the succinate dehydro-genase/fumarate reductase and the cytochrome bd complexwere downregulated for most of their components at themRNA level. The most noticeable downregulated groups ofgenes belong to the chemotaxis family, including cheA (0.46-fold), cheB (0.47-fold), cheY (0.44-fold), and cheW (0.35-fold)and the potassium transport system, including kdpA (0.40-fold), kdpB (0.41-fold), kdpC (0.47-fold), kdpD (0.33-fold), andkdpE (0.48-fold). For the genes in the kdp operon, we suspectthat the changing pattern was actually due to the upregulationof these genes in strain JS71 because the culture density washigher than that of JS7131. Consistent with this is that thedownregulation of kdpA (JS7131/JS71) is more pronounced at5 h compared to the 3 h of YidC depletion, as determined byreal-time PCR; JS71 is entering stationary phase at the 5-hgrowth time (Fig. 3). The higher density of JS71 may lower theavailable potassium ions in the medium, which in turn couldinduce the expression of the high-affinity potassium trans-porter genes.

cadA and cadB were the two most highly upregulated genesin our microarray study. cadA was upregulated 32.8-fold, whilecadB was upregulated 20.7-fold (Table 1). The cad operon isgenerally induced under acidic external pH or anoxic condi-tions. For example, cadA was shown to be upregulated underacidic conditions (15). Since YidC depletion significantly low-ers the PMF due to a perturbation in the assembly of therespiratory complexes F1Fo ATP synthase and cytochrome booxidase and affects several important genes within the protontransport pathway, the pH balance is likely to be out of control,and certain genes were activated to cope with this. We alsoused real-time PCR to confirm that cadA was highly expressedafter 5 h of depletion (Fig. 3).

YidC depletion affects cell motility. Two flagellar biosynthe-sis genes, fliS and fliC, were downregulated under YidC deple-tion conditions. To test if flagellar function was indeed af-fected, cell motility assays were carried out. Equal amounts ofJS7131 and JS71 cells (normalized to the same number ofcells) were applied to motility agar containing 0.2% glucoseand incubated at 37°C for 24 h (Fig. 7). After 24 h of incuba-tion, the diameter of the JS71 swimming zone was approxi-mately 5 times larger than that with JS7131. JS7131 growsmuch slower than JS71 under the YidC depletion condition,and eventually YidC depletion leads to cell death, so we addeda low concentration of arabinose (final concentration, 0.002%)to the plate to ensure JS7131 was kept alive during the 24-htest period. The results confirmed that YidC depletion had anegative effect on flagellar function.

Proteome analysis during the YidC depletion condition. (i)Overall regulation pattern. To understand how the cell re-sponds to YidC depletion, we analyzed the global change ofthe proteome by 2D gel electrophoresis experiments. To im-prove the resolution, we separated the inner membrane frac-tion from the soluble fraction. Equal amounts of protein fromeach fraction from both JS71 and JS7131 were applied toimmobilized pH gradient (IPG) strips. The 2D gels werestained with SYPRO ruby gel stain. Figure 8 shows 2 repre-

FIG. 7. YidC depletion affects cell motility. After growth for 5 h inLB medium supplemented with 0.2% glucose, equal numbers (as de-termined using a microscope and counting chamber) of JS71 (YidCexpressed) and JS7131 (YidC depleted) cells were applied to a motilityagar plate containing 0.2% glucose and 0.002% arabinose. This arabi-nose concentration was tested to be sufficient to keep JS7131 cells alivebut did not allow growth within 24 h. Plates were incubated at 37°C for24 h. The size of the JS7131 colony was 0.6 0.1 cm. The size of theJS71 colony was 3.2 0.3 cm. Sizes were measured independentlyusing 5 plates for each of the strains. Only one representative plate isshown here.



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sentative gels for both YidC expression and YidC depletionconditions for the soluble fraction. The membrane fraction wasnot used for a quantitative analysis since the separating reso-lution on the 2D gels was not sufficient. The protein spotsshowing different intensities under YidC depletion were se-lected, as indicated by arrows on the figure. Protein spots werecut out and analyzed by mass spectrometry. The results arelisted in Table 3. Some proteins are listed as the precursorform. This is because the database has no separate entries forpreprotein and the mature proteins, and the program used toanalyze the mass spectrometry data could not differentiatebetween a preprotein and the mature protein.

We succeeded in identifying 17 proteins in which theamounts of the proteins in the soluble fraction were changedby YidC depletion either greater than a 2-fold increase orgreater than a 2-fold decrease. The predicted molecular massand pI reasonably match the measured molecular mass and pI

for each protein. Nine proteins were found in larger amountsand 8 in smaller amounts in the YidC-depleted cells. Notably,we detected only a fraction of the known proteins on the 2Dgels. This proteomic study was carried out to identify relativelyabundant proteins with altered protein levels in the fractionswhen YidC is depleted. Our intent was not to identify exhaus-tively all of the proteins affected by YidC depletion. We con-firmed the altered levels of several proteins by Western blot-ting (Fig. 9). In addition, we showed the level of Foc is stronglyreduced in the membrane fraction when YidC is depleted (39,45) and Foa is reduced (45). MalF was also found to be de-creased when YidC is depleted, as previously reported (41).

(ii) Stress response. Universal stress protein UspA, whichhad shown a 23% upregulation on the mRNA level (see theExcel file in the supplemental material), showed a 2.7-foldincrease on the 2D gel. UspA functions to protect the cell fromDNA damage (6). This gene is upregulated when E. coli cells

FIG. 8. Comparison of the protein patterns in the soluble fractions from JS71 (control) and JS7131 (YidC depletion) cells. Two representative2D gels are shown (JS71 soluble and JS7131 soluble). Gels were stained with SYPRO ruby. The arrow indicates the spots that were cut out forMS analysis.



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are subjected to growth arrest conditions (19, 20), regardless ofthe reason for the cell stasis. Thus, we believe the level ofUspA protein is elevated because the growth of the cells isarrested after YidC depletion in JS7131.

The phage shock protein A was well resolved on the 2D gelof the membrane fraction (data not shown) and showed a13-fold increase consistent with its mRNA showing a 13-foldincrease on the gene chip. This agrees with several previousstudies which showed that phage shock protein response wasswitched on during the YidC depletion condition (12).

(iii) Proteases and chaperones. The chaperones that wereenhanced after YidC depletion on the 2D gels were GroEL(4-fold), GroES (3-fold), and ClpB (3-fold) (Fig. 8; Table 3).Since the YidC depletion has been shown to result in themisfolding of certain polytopic membrane proteins (17), theincrease in the levels of chaperones was somewhat expected.However, none of them showed significant upregulation on themRNA level (see the Excel file in the supplemental material),which could be a problem due to a poor probe design. To verifythe mRNA level, groEL and groES were subjected to real-timePCR experiments using different probes designed in our lab. Inreal-time PCR experiments, while groEL showed no significantchange, groES showed an 8.1-fold upregulation (P � 0.0006).Why gene expression of GroEL is not significantly changed butthe protein level increases is not clear; it may be that underYidC depletion conditions, the GroEL protein itself is stabi-lized.

(iv) Metabolism and transporters. YidC depletion alsoleads to changes at the protein levels of proteins and enzymesinvolved in cellular metabolism (Table 3). Likewise, the malt-ose binding protein is increased 9-fold in the soluble fraction

(which includes periplasmic and cytoplasmic proteins) whenYidC is depleted for 5 h. Exactly why this change occurs isunclear. On the other hand, the periplasmic cysteine-bindingprotein FliY is found in reduced levels in the soluble fractionon the 2D gel. This may be due to JS71 entering the earlystationary phase, causing higher expression of this well-knownstationary-phase-induced gene (see Fig. S2 in the supplementalmaterial) and leading to a relative decrease for the FliY pro-tein in the JS7131 strain, which is not in stationary phase. Thisagrees with 0.4-fold downregulation of its mRNA on the genechip. FliY acts as a cell density sensor and transfers the signalto alternative sigma factor sigma F. Since the JS71 culture isdenser than JS7131 after 5 h of growth, FliY is more likely tobe induced.

The proteomic and gene chip studies reported here showthat YidC depletion results in multiple changes in the cell atthe protein and mRNA level. These changes presumably allowthe cell in part to cope with the defects in membrane proteininsertion and folding of proteins.

DISCUSSION

The primary reason for undertaking this study was to deter-mine if there is a quality control system that is induced tohandle the stress when membrane protein assembly and fold-ing are impaired by YidC depletion. A clear role of the Cpxenvelop stress response was found. We determined also in thisstudy that the PspA induction that occurs during YidC deple-tion is largely independent of the PMF. We performed a mi-croarray analysis at 5 h so that we could evaluate the broadphysiological contribution of YidC to the bacterial cell and the

TABLE 3. List of significantly affected proteins from 2D gel experimentsa

Protein type and spot no. Protein MW pI JS7131/JS71 change inprotein level

Fold change inprotein level

Gene expression(microarray)

Metabolism2 Alkyl hydroperoxide reductase subunit C 20,862 5.03 Up 2.00 1.14 (P � 0.4)5 l-Serine dehydratase 1 49,388 5.18 Up 2.51 1.33 (P � 0.2)7 Alkyl hydroperoxide reductase, subunit F 56,487 5.47 Up 2.41 1.42 (P � 0.2)10 Enhancing lycopene biosynthesis protein 2 23,252 4.68 Down 0.45 0.85 (P � 0.01)12 Pyruvate kinase I 51,039 5.77 Down 0.50 0.72 (P � 0.9)13 Enolase 45,683 5.32 Down 0.35 0.84 (P � 0.3)15 7-�-Hydroxysteroid dehydrogenase 26,990 5.22 Down 0.43 0.28 (P � 0.0003)16 KHG/KDPG aldolaseb 22,441 5.57 Down 0.23 No exact match17 Flavohemoprotein 43,954 5.48 Down 0.27 No exact match

Protease and chaperones8 Chaperone protein ClpB 95,697 5.37 Up 3.26 0.56 (P � 0.007)9 10-kDa chaperonin GroES 10,381 5.15 Up 2.77 0.71 (P � 0.02)3 60-kDa chaperonin GroEL 57,464 4.85 Up 3.90 0.91 (P � 0.7)11 DnaK 69,130 4.83 Down 0.34 0.81 (P � 0.1)

Stress response4 Universal stress protein A 16,113 5.11 Up 2.73 1.23 (P � 0.03)

Transporter6 Maltose-binding periplasmic protein precursor 43,360 5.53 Up 9.04 2.53 (P � 0.0008)14 Cystine-binding periplasmic protein precursor 29,021 6.21 Down 0.42 0.43 (P � 0.0005)

Other1 Protein YgiW precursor 14,059 5.08 Up 3.57 0.54 (P � 0.0007)

a Significantly affected means a �2-fold increase or �2-fold decrease.b KHG, 4-hydroxy-2-oxoglutarate; KDPG, phospho-2-dehydro-3-deoxygluconate.



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considerable consequences to the cell when it is depleted. Ofcourse, this includes primary, secondary, and tertiary effects.As a primary effect, YidC-dependent substrates such as sub-units a and c of the F1Fo ATP synthase and CyoA start to beinhibited for membrane insertion after 1 h of depletion and arestrongly inhibited in membrane insertion after 3 h of depletion(J. Yuan and R. E. Dalbey, unpublished data).

In the microarray analysis, we confirmed the induction of thephage shock response during YidC depletion (12). We alsofound that the universal stress response protein A was upregu-lated in JS7131 under the YidC depletion condition. This ismost likely because of an induced growth arrest, previouslyshown to switch on the expression of this protein (18). Anotherfamily of stress response proteins shown to be highly upregu-lated is the cold shock protein Csp family, which generallyfunctions as RNA chaperones. The heat shock proteins weregenerally not affected. Of the 17 heat shock proteins that can

be analyzed by the gene chip method, only 2 of them, HtrC andDdg, showed significant upregulation (see the Excel file in thesupplemental material). However, the heat shock proteinsGroEL and GroES were found in higher abundance in thesoluble fraction when YidC was depleted. Since YidC deple-tion affects the PMF of the cell (39), we compared our mi-croarray study with YidC-depleted cells with a published mi-croarray study from another group where E. coli cells weretreated with CCCP to dissipate the proton gradient across theinner membrane (22). The two sets of micro-gene chip datawere drastically different, showing that YidC is important forphysiological processes other than to assemble the respiratorycomplexes needed to generate a PMF.

YidC depletion affects gene expression of ATP synthase andelectron transport chain complexes. Our data indicate that thethree integral membrane protein subunits of the Fo sector ofthe ATP synthase are significantly upregulated at the mRNAlevel upon YidC-depletion. Specifically, the genes encodingsubunits a, b, and c from the Fo sector of ATP synthase wereupregulated 2- to 3-fold (see the Excel file in the supplementalmaterial). Likewise, the expression of the genes encoding theF1 sector proteins was upregulated 1.5- to 3-fold (see the Excelfile in the supplemental material).

In the YidC-depleted cells, the expression of the genes en-coding CyoABCDE (cytochrome bo3 ubiquinol oxidase) wasupregulated (ranging from 2.5- to 6.8-fold) (Table 1; see theExcel file in the supplemental material). cyoB and cyoD wereselected and checked by real-time PCR, which showed 2.27-and 2.29-fold upregulation, respectively. This upregulation isreasonable as a response to assemble more cytochrome bo3

oxidase and F1FO ATP synthase complexes and to compensatefor the reduced amount of these complexes in the cell innermembrane. The identity of the signal that switches on tran-scription of the ATP synthase and cytochrome bo3 oxidaseoperons warrants further investigation. We suspect that YidC-depleted cells are unable to maintain the cytoplasmic pH dueto the impaired proton pumps. This explains the upregulatedcadA and cadB genes, which were previously shown to beinduced under the low internal cellular pH condition (16).

Along with the genes encoding F1Fo ATP synthase and cy-tochrome bo3 oxidase, other electron transport chain com-plexes also showed an interesting regulation pattern whenYidC is depleted. Genes encoding the subunits for the NADHdehydrogenase I complex in the electron transport chain wereupregulated: nuoABCEFGHIJKLMN showed 1.7 to 4.9-foldupregulation (see the Excel file in the supplemental material).For real-time PCR analysis, nuoB and nuoM were selected,and we found 3.19- and 2.96-fold upregulation, respectively.We suspect the NADH dehydrogenase II complex that nor-mally operates under aerobic conditions is perturbed by theYidC depletion and complex I is induced instead. At the sametime, genes encoding the subunits for the fumarate reductasecomplex were downregulated. Specifically, genes frdABCD offumarate reductase were downregulated 0.2- to 0.4-fold (seebelow). Interestingly, it has recently been shown that FrdA isstrongly affected by YidC depletion of anaerobically growingE. coli (24).

Our hypothesis is that the inhibition in the assembly ofelectron transport chain complexes likely affects the redox bal-ance inside YidC-depleted cells, causing quinols and oxygen to

FIG. 9. Western blot analysis of selected proteins. To verify the 2Dgel results, protein samples were collected from cell lysates derivedfrom JS7131 and JS71 after 5 h of growth in LB medium plus 0.2%glucose and subjected to Western blot analysis using the indicatedantibodies. YidC, phage shock protein PspA, MBP, and the chaper-ones DnaJ and GroEL were analyzed. In the case of MalF and the FoATPase subunits, it was necessary to enrich the proteins by isolatingthe membranes. Inner membrane vesicles from JS7131 and JS71 underthe 5-h YidC depletion condition were purified and analyzed by West-ern blotting using antibodies against Foa, Fob, Foc, and MalF.



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accumulate in the cell. To verify this hypothesis, we firstchecked the fumarate nitrate reductase (Fnr) pathway, whichis one of the major regulatory pathways used to handle redoxbalance problems. Fnr is an O2-sensing transcriptional regula-tor, which can activate or inactivate transcription of certaingenes when low oxygen levels are present. Under these condi-tions, Fnr can form dimers and thereby bind their promoterregions. For the frdABCD, aspA, fumB, and pfl genes, Fnr actsas an activator at low oxygen levels (11, 31, 33). Thus, in thepresence of oxygen, the transcription level is low in all of thesegenes. In our data set, frdABCD genes were downregulatedand also pfl was slightly downregulated. In the presence ofoxygen, an increase in the transcription level of cyoABCD andsdhABCD genes had been reported (3, 21). Our results showthat the expression of the cyoABCD genes was upregulated andthe expression of sdhABCD genes was slightly upregulatedwhen YidC is depleted, indicating that sufficient oxygen wasavailable and Fnr was not functioning as a repressor. Our dataagree well with data reported in a previous study which used agene chip method to study the effects of oxygen availability andFnr pathway. For a total of 36 genes analyzed in that paperwhich were claimed to use Fnr dimer as a repressor, the ex-pression level is expected to be elevated when oxygen ispresent (see Table S5 in the supplemental material) (27). In-deed, 18 of these genes were upregulated according to ourgene chip data, while others either were not found in ouranalysis or were not affected (see the Excel file in the supple-mental material). For a total of 58 genes which were claimed touse Fnr dimer as an activator (see Table S6 in the supplemen-tal material) (27), the expression level should be maintained ata normal level in our experiment, where more oxygen ispresent. We found that 47 of these genes showed no significantchange, 2 of them were not found, 7 were upregulated, and 2were downregulated (see the Excel file in the supplementalmaterial). We conclude that oxygen is not limiting under YidCdepletion conditions.

The high-level induction of the psp operon under the YidC-depleted condition is primarily not due to the disturbance ofthe PMF. PspA is a highly expressed protein when YidC isdepleted in the cell (12, 39). The induction of pspA in YidC-depleted cells was thought to be the result of an impaired PMFbecause YidC depletion leads to a decrease in the PMF (39)and pspA was also induced after E. coli K561 cells were ex-posed to 40 or 60 �M CCCP (42). It is well established thatCCCP, a protonophore, collapses the proton gradient acrossthe inner membrane. Kleerebezem et al. had shown that thePspA protein helps to maintain the PMF when a mutant PhoEprotein is overexpressed and makes the membrane leaky (13).However, our data show that CCCP treatment only leads to a2- to 4-fold induction of pspA, compared to 30-fold pspA in-duction when YidC is depleted (Fig. 6). Also, we noticed thatincreasing the CCCP concentrations from 1.25 to 25 �M didnot lead to an increase of PspA. Additionally, we found thatthe high concentration of CCCP (40 �M) that Weiner andModel used for their study had a much stronger effect onprotein export and growth of the cell than YidC depletion (Fig.6) (42).

Our data are supported by another gene chip study in whichthe authors showed that CCCP (40 �M) did not activate by2-fold the phage shock response operon (22; Volker Wendisch,

University of Muenster, personal communication). In this pre-vious gene chip study, CCCP was added for 3 h of growth at afinal CCCP concentration of 40 �M. While we realize one hasto be careful comparing different microarray studies (wheredifferent growth media and genetic backgrounds of the strainare used), we find it intriguing that the overall pattern ofregulated genes was completely different from what we ob-served for the YidC-depleted cells (22; V. Wendisch, personalcommunication). For example, among 27 genes which werehighly upregulated (�5-fold) in the CCCP-treated cells, onlyone of them was found to be upregulated in our YidC deple-tion study (see Table S4 in the supplemental material). For the10 genes which were highly downregulated (�30%) in CCCP-treated cells, none was found to be downregulated in the YidC-depleted cells in our study. Strikingly, the phage shock operonwas not measurably induced upon CCCP treatment (22; V.Wendisch, personal communication), while YidC depletioncaused significant induction of phage shock proteins. There-fore, we conclude that PspA is strongly induced when mem-brane protein insertion is perturbed to compensate for theproblems that result when the membrane is damaged, asidefrom a drop in the PMF. The most likely scenario is thatmultiple factors activate the psp operon once YidC is depleted,and it will be interesting to determine what factors are involvedin this activation.

Quality control system for bacterial membrane protein. Wefound in our microarray study that the expression of severalprotease and chaperone genes was upregulated, possibly tocope with the defects in membrane protein insertion, folding,and assembly when the YidC level is reduced (see the Excel filein the supplemental material). This includes the genes encod-ing peptidyl prolyl cis-trans isomerase A, protease III (a car-boxypeptidase), DnaJ, FimC, and trigger factor. In additionthe levels of ClpB, GroEs, and GroEL proteins in the solublefraction were increased when YidC was depleted. Surprisingly,expression of a number of inner membrane proteases such asFtsH, GlpG, HtpX, YaeL, and YfbL (see the Excel file in thesupplemental material for the complete list) were upregulatedto only a small extent (140% to 192% compared to control).Since there is no obvious inner membrane protease specificallyinvolved in handling protein stress due to misfolding of mem-brane proteins, the quality control of inner membrane proteinsis most likely handled by several proteases.

Our results support the hypothesis that the Cpx pathwayplays a role in dealing with improper membrane protein bio-genesis, in addition to its well-established functions in handlingaltered lipid composition, overproduction of the outer mem-brane lipoprotein, and high pH (25). We observed upregula-tion of dsbA and ppiA genes from our gene chip study (see theExcel file in the supplemental material) and the cpxP genefrom real-time PCR data. We compared our gene chip datawith data from another study which identified 26 genes that areregulated by the Cpx pathway (see Table S1 in the supplemen-tal material) (7). Eleven of the genes showed a similar regu-lation pattern in our study, and 2 of them were not found, whileothers were either not significantly affected or did not givereliable data. While not all of the Cpx-regulated genes areactivated in the YidC depletion study, there is still good agree-ment. Importantly, current studies support the notion thatCpxP (which is upregulated 15-fold) can only be induced by the



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Cpx pathway (T. Silhavy, personal communication). Therefore,we conclude that the Cpx stress pathway is activated by YidCdepletion, and this supports a previous study that proposed aCpx activation after YidC is depleted based on cpxP andrpoHP3 promoter LacZ fusions (32).

Our data clearly do not support the activation of the sigmaE pathway, as previously proposed (32). For sigma E pathway-controlled genes, among 31 transcription units, which wereshown to be regulated by the sigma E factor with the conservedpromoter motif upstream of the genes (see Table S2 in thesupplemental material) (26), 14 of them showed a sigma E-controlled regulation pattern, 4 of them showed the oppositeregulation pattern, and 13 of them did not show a significantchange. Also, rpoE mRNA is usually a good indicator of sigmaE activity, and there was no change in our microarray data.Therefore, no significant correlation was found in this analysis.

Conclusion. In this study, the global pattern of gene expres-sion at the mRNA and protein level was obtained and analyzedunder YidC depletion conditions in E. coli. We demonstratedthat the motility of E. coli cells is impaired and the cell mor-phology is altered when YidC is depleted. Our data show thatYidC depletion affects various bacterial electron transportchain complexes, and their genes are upregulated to handlethese problems. When analyzing the stress response caused byYidC depletion, we found that the Cpx pathway is inducedupon depletion of YidC. Surprisingly, our CCCP experimentssuggested that the phage shock response that is induced byYidC depletion is not directly the result of the partially col-lapsed PMF.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health grantGM63862 to R.E.D. and by DFG grant Ku749/6-1.

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Global Change of Gene Expression and Cell Physiology in YidC-Depleted Escherichia coli

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